High D dimers and low global fibrinolysis coexist in COVID19 patients: what is going on in there?

BACKGROUD: COVID-19 coagulopathy linked to increased D-dimer levels has been associated with high mortality (Fei Z et al. in Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet (London, England) 395(10229):1054-62, 2020). While D-dimer is accepted as a disseminated intravascular coagulation marker, rotational thromboelastometry (ROTEM) also detects fibrinolysis (Wright FL et al. in Fibrinolysis shutdown correlates to thromboembolic events in severe COVID-19 infection. J Am Coll Surg (2020). Available from https://pubmed.ncbi.nlm.nih.gov/32422349/ [cited 14 Jun 2020]; Schmitt FCF et al. in Acute fibrinolysis shutdown occurs early in septic shock and is associated with increased morbidity and mortality: results of an observational pilot study. Ann Intensive Care 9(1):19, 2019). We describe the ROTEM profile in severely ill COVID-19 patients and compare it with the standard laboratory coagulation test. METHODS: Adult patients diagnosed with COVID-19 admitted to the ICU were prospectively enrolled after Ethics Committee approval (HCB/2020/0371). All patients received venous thromboembolism prophylaxis; those on therapeutic anticoagulation were excluded. The standard laboratory coagulation test and ROTEM were performed simultaneously at 24-48 h after ICU admission. Sequential organ failure assessment (SOFA), disseminated intravascular coagulation (DIC) and sepsis-induced coagulopathy (SIC) scores were calculated at sample collection. RESULTS: Nineteen patients were included with median SOFA-score of 4 (2-6), DIC-score of 1 (0-3) and SIC-score of 1.8 (0.9). Median fibrinogen, D-dimer levels and platelet count were 6.2 (4.8-7.6 g/L), 1000 (600-4200 ng/ml) and 236 (136-364 109/L), respectively. Clot firmness was above the normal range in the EXTEM and FIBTEM tests while clot lysis was decreased. There was no significant correlation between ROTEM or D-dimer parameters and the SOFA score. CONCLUSION: In COVID-19 patients, the ROTEM pattern was characterized by a hypercoagulable state with decreased fibrinolytic capacity despite a paradoxical increase in D-dimer levels. We suggest that, in COVID-19 patients, the lungs could be the main source of D-dimer, while a systemic hypofibrinolytic state coexists. This hypothesis should be confirmed by future studies.

Clinical characterization of gingival type of burning mouth syndrome: a cross-sectional study.

BACKGROUND: The aim of this study is to investigate the prevalence of localized intraoral neuropathic pain in a cluster of patients who reported the involvement of gingival site as only clinical manifestation of dysesthesia, analysing type and distribution of symptoms. MATERIAL AND METHODS: Burning mouth syndrome (BMS) patients were enrolled in the study. Patients were screened through laboratory test and a conventional oral examination with periodontal chart. A questionnaire to collect data on symptoms, oral site involved, quality of sleep, anxiety was submitted to all the patients. RESULTS: A total of 236 patients were recruited. Seventy-six patients (32.2%) presented generalized type, whereas 160 (67.8%) had localized type. In the localized BMS group, the gingiva was involved in 70 patients and in 33 of these it was the only site involved. In the gingival subgroup, 35 patients reported burning, 29 pain and 24 xerostomia. CONCLUSIONS: To best our knowledge, this study is the first that analyses gingival involvement as the only site in BMS and it could encourage further investigations to understand the etiopathogenesis of gingival BMS.

Treatment of a peri-implantitis defect using a mini-invasive surgical approach (MISA) and deproteinized bovine bone mineral with 10% collagen: a case series.

This pilot study evaluates the effectiveness of the Mini-Invasive Surgical Approach (MISA) in the treatment of peri-implantitis defect. MISA is based on the use of the deproteinized bovine bone mineral with 10% collagen in combination with a minimal flap. The main principle is the elevation of a flap to access to the peri-implantitis defect only on one side (palatal aspect), leaving the opposite site intact. The study was designed as a non-controlled, non-randomized pilot study. In 10 consecutive subjects, 10 implants with diagnosis of perimplantitis were selected. In all the treated sites, primary closure was obtained at completion of the surgical procedure. No relevant pain, edema and hematoma were noted. The reported surgical approach resulted in significant clinical and radiographic improvements while limiting patient morbidity. After 1-year follow-up minimal mucosal recession, probing depth reduction and radiographic bone gain were noted.

Cortical responses before 6 months of life associate with later autism.

Autism spectrum disorder (ASD) is a common, highly heritable, developmental disorder and later-born siblings of diagnosed children are at higher risk of developing ASD than the general population. Although the emergence of behavioural symptoms of ASD in toddlerhood is well characterized, far less is known about development during the first months of life of infants at familial risk. In a prospective longitudinal study of infants at familial risk followed to 36 months, we measured functional near-infrared spectroscopy (fNIRS) brain responses to social videos of people (i.e. peek-a-boo) compared to non-social images (vehicles) and human vocalizations compared to non-vocal sounds. At 4-6 months, infants who went on to develop ASD at 3 years (N = 5) evidenced-reduced activation to visual social stimuli relative to low-risk infants (N = 16) across inferior frontal (IFG) and posterior temporal (pSTS-TPJ) regions of the cortex. Furthermore, these infants also showed reduced activation to vocal sounds and enhanced activation to non-vocal sounds within left lateralized temporal (aMTG-STG/pSTS-TPJ) regions compared with low-risk infants and high-risk infants who did not develop ASD (N = 15). The degree of activation to both the visual and auditory stimuli correlated with parent-reported ASD symptomology in toddlerhood. These preliminary findings are consistent with later atypical social brain responses seen in children and adults with ASD, and highlight the need for further work interrogating atypical processing in early infancy and how it may relate to later social interaction and communication difficulties characteristic of ASD.

Correlation between plasma fibrinogen and FIBTEM thromboelastometry during liver transplantation: a comprehensive assessment.

BACKGROUND: Thromboelastometry may reduce red blood cell (RBC) transfusion in liver transplantation (LT). Fibrinogen concentration is a primary determinant of FIBTEM maximum clot firmness (MCF), but several factors could affect the correlation between FIBTEM MCF and fibrinogen values. We aimed to investigate (1) the concordance between fibrinogen level and FIBTEM MCF and (2) the association of fibrinogen level and FIBTEM MCF with RBC transfusion during LT. METHODS: A post hoc analysis of data from a randomized, multicentre, double-blind, saline/fibrinogen trial was used (NCT01539057). A total of 86 adult patients were included. RESULTS: The Lin concordance coefficient (LCC) between FIBTEM MCF and fibrinogen levels with the model formula 1·3679 + 0·05414* FIBTEM MCF was poor overall (LLC [95% CI]: 0·387 [0·340 to 0·432]) and moderate for the preperfusion period (LLC [95% CI]: 0·789 [0·747 to 0·824]), but very poor for the postreperfusion period (LLC [95% CI] 0·170 [0·105 to 0·233]). The model assessed for RBC transfusion for FIBTEM MCF showed an area under the curve of 0·788 [0·745-0·832]. Patients with FIBTEM MCF values <8 mm had a significantly higher likelihood of receiving RBC than patients with higher values. (OR [95% CI]: 2·08 [1·30-3·33], P = 0·002). FIBTEM MCF values over 10 mm do not appear to reduce the likelihood of RBC transfusion. CONCLUSION: FIBTEM MCF is not a good indicator of plasma fibrinogen values after graft reperfusion. FIBTEM MCF >8 mm during the LT procedure is associated with less RBC transfusion. FIBTEM MCF values over 10 mm could lead to unnecessary fibrinogen administration.

Are the school prevention programmes - aimed at de-normalizing smoking among youths - beneficial in the long term? An example from the Smoke Free Class Competition in Italy.

Tobacco smoking by young people is of great concern because it usually leads to regular smoking, nicotine addiction and quitting difficulties. Young people "hooked" by tobacco maintain the profits of the tobacco industry by replacing smokers who quit or die. If new generations could be tobacco-free, as supported by tobacco endgame strategies, the tobacco epidemic could end within decades. Smoking prevention programmes for teens are offered by schools with the aim to prevent or delay smoking onset. Among these, the Smoke Free Class Competition (SFC) was widely implemented in Europe. Its effectiveness yielded conflicting results, but it was only evaluated at short/medium term (6 - 18 months). The aim of this study is to evaluate its effectiveness after a longer follow-up (3 to 5 years) in order to allow enough time for the maturing of the students and the internalization of the experience and its contents. Fifteen classes were randomly sampled from two Italian high schools of Bologna province that regularly offered the SFC to first year students; 382 students (174 participating in the SFC and 208 controls) were retrospectively followed-up and provided their "smoking histories". At the end of their last year of school (after 5 years from the SFC), the percentage of students who stated that they were regular smokers was lower among the SFC students than in controls: 13.5% vs 32.9% (p=0.03). From the students' "smoking histories", statistically significant protective ORs were observed for SFC students at the end of 1st and 5th year: 0.42 (95% CI 0.19-0.93) and 0.32 (95% CI 0.11-0.91) respectively. Absence of smokers in the family was also a strongly statistically significant factor associated with being a non-smoker student. These results suggest that SFC may have a positive impact on lowering the prevalence of smoking in the long term (5 years).

Impact of Preemptive Fibrinogen Concentrate on Transfusion Requirements in Liver Transplantation: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

We hypothesized that preemptive fibrinogen administration to obtain an initial plasma level of 2.9 g/L would reduce transfusion requirements in liver transplantation. A randomized, multicenter, hemoglobin-stratified, double-blind, fibrinogen-versus-saline-controlled trial was conducted. The primary end point was the percentage of patients requiring red blood cells. We evaluated 51 patients allocated to fibrinogen and 48 allocated to saline; the primary end point was assessed using data for 92 patients because the electronic record forms were offline for three patients in the fibrinogen group and four in the saline group. We injected a median of 3.54 g fibrinogen preemptively in the fibrinogen group. Nine patients in the saline group (20.9%) required fibrinogen at graft reperfusion (compared with one patient [2.1%] in the fibrinogen group; p = 0.005). Blood was transfused to 52.9% (95% confidence interval [CI] 42.5-63.3%) in the fibrinogen group and 42.74% (95% CI 28.3-57.2%) in the saline group (p = 0.217). Relative risk for blood transfusion was 0.80 (95% CI 0.57-1.13). Thrombotic events occurred in one patient (2.1%) and five patients (11.4%) in the fibrinogen and saline groups, respectively. Seven patients (14.6%) in the fibrinogen group and nine (20.3%) in the saline group required reoperation. Preemptive administration of fibrinogen concentrate did not influence transfusion requirements.

Liver Transplant From Unexpected Donation After Circulatory Determination of Death Donors: A Challenge in Perioperative Management.

Unexpected donation after circulatory determination of death (uDCD) liver transplantation is a complex procedure, in particular when it comes to perioperative recipient management. However, very little has been published to date regarding intraoperative and immediate postoperative care in this setting. Herein, we compare perioperative events in uDCD liver recipients with those of a matched group of donation after brain death liver recipients. We demonstrate that the former group of recipients suffers significantly greater hemodynamic instability and derangements in coagulation following graft reperfusion. Based on our experience, we recommend a proactive recipient management strategy in uDCD liver transplantation that involves early use of vasopressor support; maintaining adequate intraoperative levels of red cells, platelets, and fibrinogen; and routinely administering tranexamic acid before graft reperfusion.

Coagulation profile after plasma exchange using albumin as a replacement solution measured by thromboelastometry.

BACKGROUND: Significant decrease in fibrinogen and other coagulation proteins is observed after plasma exchange when albumin is used as a replacement fluid. Little is known about how those changes impact on thromboelastometry (TEM). The aim of this study was to describe the changes in TEM after performing plasma exchange procedures using 5% albumin as a replacement fluid and its correlation with the standard coagulation tests. STUDY DESIGN AND METHODS: Consecutive patients treated with plasma exchange were prospectively recorded. We performed laboratory and TEM assays extrinsec patway [EXTEM and fibrin-based TEM (fibTEM)] from patient's blood sampled immediately before starting and immediately after finishing each procedure. Bleeding events during and after the procedure were monitored. Mann-Whitney U-test and Spearman's correlation test were used when needed. RESULTS: After plasma exchange, all TEM parameters decreased under normal limits, especially the clotting time (CT): 62% reduction (range: 35-84%) and the fibTEM: 50% (range: 62-27%); maximum clot firmness (MCF) decreased to a lesser extent, 23% (range: 29-21%). Main TEM parameters showed a strong correlation with fibrinogen values: CT, r = -0·81; MCF, r = 0·85; and fibTEM, r = 0·79. No bleeding complications were observed. CONCLUSION: A profound derangement of TEM parameters was observed after PE, evidenced by delay in the clot formation and reduction in the clot firmness. More studies are warranted to elucidate the clinical implications of derangement in TEM parameters in patients without concomitant coagulopathy.

Rotation thromboelastometry velocity curve predicts blood loss during liver transplantation.

BACKGROUND: Patients undergoing liver transplantation (LT) have a high risk of bleeding. The goal of this study was to assess whether the first derivative of the velocity waveform (V-curve) generated by whole blood rotation thromboelastometry (ROTEM®) can predict blood loss during LT. METHODS: Preoperative V-curve parameters were retrospectively evaluated in 198 patients. Patients were divided into quartiles based on blood loss: low (LBL) in the first quartile and high (HBL) in the higher quartiles. A subgroup analysis was performed with patients stratified according to cirrhosis aetiology. A logistic regression model and receiver operator characteristics (ROC) curve were used to test the capacity of the V-curve, to discriminate between LBL and HBL. RESULTS: In the HBL group, the V-curve showed a lower maximum velocity of clot generation (MaxVel), a lower area under maximum velocity curve (AUC), and a higher time-to-maximum velocity (t-MaxVel) than in the LBL group. t-MaxVel was the only parameter showing a capacity to discriminate between the two groups, with a ROC area of 0.69 (95% CI; 0.62-0.74). The ROC area was 0.78 (95% CI; 0.75-0.83) for the 148 patients with cirrhosis, 0.73 (0.60-0.82) for patients with viral hepatitis and 0.83 (0.78-0.96) for patients with alcoholic hepatitis, the group that showed the best discriminative capacity. Moderate but significant correlations were found between all parameters of V-curve and BL. CONCLUSIONS: Pre-transplant V-curve obtained from ROTEM is a promising tool for predicting BL risk during LT, particularly in patients with cirrhosis.

Steam pretreatment of Saccharum officinarum L. bagasse by adding of impregnating agents for advanced bioethanol production.

The main byproduct of the sugarcane industry, Saccharum officinarum L. bagasse (sugarcane bagasse, SCB), is widely used as lignocellulose biomass for bio-ethanol (EtOH) production. In this research study, SCB was pretreated by steam explosion (SE) method using two different impregnating agents: sulfur dioxide (SD) and hydrogen peroxide (HP). As matter of fact, the use of impregnating agents improves the performance of SE method, increasing the concentrations of fermentable sugars after enzymatic saccharification, and decreasing the inhibitor compounds produced during the steam pretreatment step. The aim of this study was to investigate and compare the use of the two impregnating agents in various SE-conditions in order to optimize pretreatment parameters. For every pretreatment condition, it has been evaluated: concentration of fermentable sugars, glucose and xylose yields, and the effects of the inhibitor compounds on enzymatic hydrolysis step. The obtained results allow to improve the efficiency of the whole process of bio-EtOH synthesis enhancing the amount of fermentable sugars produced and the eco-sustainability of the whole process. Indeed, the optimization of steam pretreatment leads to a reduction of energy requirements and to a lower environmental impact.

Enamel matrix derivative and bone grafts for periodontal regeneration of intrabony defects. A systematic review and meta-analysis.

OBJECTIVE: The aim of the present systematic review and meta-analysis was to assess the clinical efficacy of regenerative periodontal surgery of intrabony defects using a combination of enamel matrix derivative (EMD) and bone graft compared with that of EMD alone. MATERIALS AND METHODS: The Cochrane Oral Health Group specialist trials, MEDLINE, and EMBASE databases were searched for entries up to February 2014. The primary outcome was gain of clinical attachment (CAL). Weighted means and forest plots were calculated for CAL gain, probing depth (PD), and gingival recession (REC). RESULTS: Twelve studies reporting on 434 patients and 548 intrabony defects were selected for the analysis. Mean CAL gain amounted to 3.76 ± 1.07 mm (median 3.63 95 % CI 3.51-3.75) following treatment with a combination of EMD and bone graft and to 3.32 ± 1.04 mm (median 3.40; 95 % CI 3.28-3.52) following treatment with EMD alone. Mean PD reduction measured 4.22 ± 1.20 mm (median 4.10; 95 % CI 3.96-4.24) at sites treated with EMD and bone graft and yielded 4.12 ± 1.07 mm (median 4.00; 95 % CI 3.88-4.12) at sites treated with EMD alone. Mean REC increase amounted to 0.76 ± 0.42 mm (median 0.63; 95 % CI 0.58-0.68) at sites treated with EMD and bone graft and to 0.91 ± 0.26 mm (median 0.90; 95 % CI 0.87-0.93) at sites treated with EMD alone. CONCLUSIONS: Within their limits, the present results indicate that the combination of EMD and bone grafts may result in additional clinical improvements in terms of CAL gain and PD reduction compared with those obtained with EMD alone. The potential influence of the chosen graft material or of the surgical procedure (i.e., flap design) on the clinical outcomes is unclear. CLINICAL RELEVANCE: The present findings support the use of EMD and bone grafts for the treatment of intrabony periodontal defects.

Reduced neural sensitivity to social stimuli in infants at risk for autism.

In the hope of discovering early markers of autism, attention has recently turned to the study of infants at risk owing to being the younger siblings of children with autism. Because the condition is highly heritable, later-born siblings of diagnosed children are at substantially higher risk for developing autism or the broader autism phenotype than the general population. Currently, there are no strong predictors of autism in early infancy and diagnosis is not reliable until around 3 years of age. Because indicators of brain functioning may be sensitive predictors, and atypical social interactions are characteristic of the syndrome, we examined whether temporal lobe specialization for processing visual and auditory social stimuli during infancy differs in infants at risk. In a functional near-infrared spectroscopy study, infants aged 4-6 months at risk for autism showed less selective neural responses to social stimuli (auditory and visual) than low-risk controls. These group differences could not be attributed to overall levels of attention, developmental stage or chronological age. Our results provide the first demonstration of specific differences in localizable brain function within the first 6 months of life in a group of infants at risk for autism. Further, these differences closely resemble known patterns of neural atypicality in children and adults with autism. Future work will determine whether these differences in infant neural responses to social stimuli predict either later autism or the broader autism phenotype frequently seen in unaffected family members.

Use of viscoelastic tests in the principle bleeding scenarios in Spanish hospitals.

Viscoelastic tests are designed to study the dynamics of clot formation, identify coagulopathies in real time, arrive at a diagnosis, and guide patient-specific administration of haemostatics. They are mainly used to treat clinically significant bleeding in any setting, and are also used in other situations involving clinically relevant alterations in haemostasis, such as coagulopathy in critically ill patients. These tests are administered following evidence-based algorithms that vary depending on the clinical context. This review summarises the results of a survey conducted in several hospitals to determine the prevalence and standardisation of viscoelastic tests in cardiac surgery, liver transplantation, and multiple trauma patients in Spain. The results reveal divergent opinions on key aspects, ranging from the diagnostic capacity of these tests to the interpretation of the basic parameters. On the basis of these findings, we propose a number of potential areas in which further research will improve the performance of these tests.

[Effectiveness of thromboprophylaxis with low molecular weight heparin in critically ill patients with COVID-19. An observational prospective, multicenter study]. / Eficacia de la tromboprofilaxis con heparina de bajo peso molecular en pacientes críticos con COVID-19: estudio observacional, prospectivo y multicéntrico.

Introduction: COVID-19 induces coagulopathy associated with an increase of thromboembolic events. Due to the lack of agreement on recommendations for thromboprophylactic management, the aim of this study was to study the dosages of LMWH used in critically ill COVID-19 patients assessing the effect on their outcome. Metohds: We evaluated data of the Reg-COVID19. According to LMWH dose two groups were analyzed: prophylaxis and treatment. Primary outcome was the relationship of LMWH dosage with mortality. Secondary outcomes included the incidence of thrombotic and bleeding events, length of ICU stay, invasive mechanical ventilation, and thrombotic and inflammatory parameters. Results: Data of 720 patients were analyzed, 258 in the prophylaxis group and 462 in the treatment group. C Reactive Protein, invasive mechanical ventilation, tocilizumab and corticosteroid treatments were related with the choice of LMWH dose. Hemorrhagic events (66/720, 9.2%) and thrombotic complications (69/720, 9.6%) were similar in both groups (P=.819 and P=.265), as was the time course of the thrombotic events, earlier than hemorrhagic ones (9 [3-18] and 12 [6-19] days respectively). Mortality was lower in prophylaxis group (25.2% versus 35.1%), but once an inverse probability weighting model was applied, we found no effect of LMWH dose. Conclusion: We found no benefit or harm with the administration of therapeutic or prophylactic LMWH dose in COVID19 critically ill patients. With a similar rate of hemorrhagic or thrombotic events, the LMWH dose had no influence on mortality. More studies are needed to determine the optimal thromboprophylaxis protocol for critically ill patients.

Effectiveness of thromboprophylaxis with low molecular weight heparin in critically ill patients with COVID-19. An observational prospective, multicenter study.

INTRODUCTION: COVID-19 induces coagulopathy associated with an increase of thromboembolic events. Due to the lack of agreement on recommendations for thromboprophylactic management, the aim of this study was to study the dosages of LMWH used in critically ill COVID-19 patients assessing the effect on their outcome. METHODS: We evaluated data of the Reg-COVID19. According to LMWH dose two groups were analyzed: prophylaxis and treatment. Primary outcome was the relationship of LMWH dosage with mortality. Secondary outcomes included the incidence of thrombotic and bleeding events, length of ICU stay, invasive mechanical ventilation, and thrombotic and inflammatory parameters. RESULTS: Data of 720 patients were analyzed, 258 in the prophylaxis group and 462 in the treatment group. C Reactive Protein, invasive mechanical ventilation, tocilizumab and corticosteroid treatments were related with the choice of LMWH dose. Hemorrhagic events (66/720, 9.2%) and thrombotic complications (69/720, 9.6%) were similar in both groups (p = .819 and p = .265), as was the time course of the thrombotic events, earlier than hemorrhagic ones (9 [3-18] and 12 [6-19] days respectively). Mortality was lower in prophylaxis group (25.2% versus 35.1%), but once an inverse probability weighting model was applied, we found no effect of LMWH dose. CONCLUSION: We found no benefit or harm with the administration of therapeutic or prophylactic LMWH dose in COVID19 critically ill patients. With a similar rate of hemorrhagic or thrombotic events, the LMWH dose had no influence on mortality. More studies are needed to determine the optimal thromboprophylaxis protocol for critically ill patients.

[An established anesthesia team-care model: over 12000 cases in a digestive endoscopy unit]. / Consolidación de un modelo asistencial basado en un equipo de anestesia: experiencia en más de 12.000 casos en una Unidad de Endoscopia Digestiva.

BACKGROUND AND OBJECTIVE: The growing demand for digestive and other endoscopic procedures outside the operating room, both in terms of type of endoscopy and number of patients, requires reorganization of the anesthesiology department's workload. We describe 2 years of our hospital digestive endoscopy unit's experience with a now well-established care model involving both anesthesiologists and nurse anesthetists. MATERIAL AND METHODS: After previously reviewing the medical records of outpatients and conducting a telephone interview about state of health, nurse anesthetists administered a combination of propofol and remifentanil through a target-controlled infusion system under an anesthesiologist's direct supervision. RESULTS: The ratio of anesthesiologists to nurses ranged from 1:2 to 1:3 according to the complexity of the examination procedure. Over 12000 endoscopies (simple to advanced) in a total of 11853 patients were performed under anesthesia during the study period. Airway management maneuvers were required by 4.9% of the patients; 0.18% required bag ventilation for respiratory depression, and 0.084% required bolus doses of a vasopressor to treat hypotension or atropine to treat bradycardia. The procedure had to be halted early in 9 patients (0.07%). No patient required orotracheal intubation and none died. Nor were any complications related to sedation recorded. CONCLUSION: The results suggest that this care model can safely accommodate a large caseload in anesthesia at an optimum level of quality.

Dysfunctional DC subsets in RCC patients: ex vivo correction to yield an effective anti-cancer vaccine.

Dendritic cells (DCs) are potent antigen-presenting cells responsible for the activation and functional polarization of specific T cells. In patients with renal cell carcinoma (RCC) and other cancers, coordinate DC and T cell defects have been reported. In particular, DC and T cell functional subsets that are not conducive to tumor clearance are hypothesized to predominate in patients with advanced-stage disease. Two major peripheral blood DC subsets have been identified in humans: myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) that are believed to mediate contrasting effects on cancer immunity. Given the lack of information regarding DC subsets in patients with RCC, in the present study we have investigated the comparative frequencies and activation states of mDC and pDC in peripheral blood, cancer tissues and lymph nodes of patients with RCC using flow cytometry and immunohistochemistry. Three monoclonal antibodies (mAbs) reactive against specific DC subsets (BDCA-2 or BDCA-4 for pDC and BDCA-1 and BDCA-3 which represent two distinct subsets of mDC, mDC1 and mDC2, respectively) were employed. We observed a significant reduction of both DC subsets in the peripheral blood of patients as compared to normal donors. Similarly, both mDC and pDC were recruited in large numbers into RCC tumor tissues, where they displayed an immature phenotype (DC-LAMP(-)) and appeared unable to differentiate into mature DC (CD83(+)) that were competent to migrate to draining lymph nodes. However, we were readily able to generate ex vivo mDC from RCC patients. These DC stimulated robust anti-tumor CTL in vitro and would be envisioned for use in DC-based vaccines applied in patients with RCC whose existing immune system is judged dysfunctional, anergic or prone to undergo apoptosis.

Regulation of group II metabotropic glutamate receptors by G protein-coupled receptor kinases: mGlu2 receptors are resistant to homologous desensitization.

We examined the regulation of mGlu2 and mGlu3 metabotropic glutamate receptor signaling prompted by the emerging role of these receptor subtypes as therapeutic targets for psychiatric disorders, such as anxiety and schizophrenia. In transfected human embryonic kidney 293 cells, G-protein-coupled receptor kinase (GRK) 2 and GRK3 fully desensitized the agonist-dependent inhibition of cAMP formation mediated by mGlu3 receptors. In contrast, GRK2 or other GRKs did not desensitize the cAMP response to mGlu2 receptor activation. Desensitization of mGlu3 receptors by GRK2 required an intact kinase activity, as shown by the use of the kinase-dead mutant GRK2-K220R or the recombinant GRK2 C-terminal domain. Overexpression of beta-arrestin1 also desensitized mGlu3 receptors and did not affect the cAMP signaling mediated by mGlu2 receptors. The difference in the regulation of mGlu2 and mGlu3 receptors was signal-dependent because GRK2 desensitized the activation of the mitogen-activated protein kinase pathway mediated by both mGlu2 and mGlu3 receptors. In vivo studies confirmed the resistance of mGlu2 receptor-mediated cAMP signaling to homologous desensitization. Wild-type, mGlu2(-/-), or mGlu3(-/-) mice were treated intraperitoneally with saline or the mixed mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]-exhane-4,6-dicarboxylic acid (LY379268; 1 mg/kg) once daily for 7 days. Inhibition of forskolin-stimulated cAMP formation by LY379268 was measured in cortical slices prepared 24 h after the last injection. Agonist pretreatment fully desensitized the cAMP response in wild-type and mGlu2(-/-) mice but had no effect in mGlu3(-/-) mice, in which LY379268 could only activate the mGlu2 receptor. We predict the lack of tolerance when mixed mGlu2/3 receptor agonists or selective mGlu2 enhancers are used continually in patients.